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Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes

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Abstract

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic β-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of β-strands arranged in supernumerary β-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary β-strands were introduced by site directed mutagenesis into the sequence encoding the β-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type β-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).

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Abbreviations

CD:

Circular dichroism spectroscopy

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

UV:

Ultraviolet

NMR:

Nuclear magnetic resonance

PCR:

Polymerase chain reaction

PNPG:

p-Nitrophenyl-β-D-glucopyranoside

PDB:

Protein data bank

TIM:

Triose-phosphate isomerase

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Acknowledgements

This work was supported by grants BIO2000-1279-C02-01 and -02. We thank Maria Ciaramella and Mosé Rossi for generous gift of plasmid pDAF1 containing the S. solfataricus lacS gene. We also thank Andrew MacCabe for critical reading of the manuscript.

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Correspondence to Julio Polaina.

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León, M., Isorna, P., Menéndez, M. et al. Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes. Protein J 26, 435–444 (2007). https://doi.org/10.1007/s10930-007-9083-2

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